Sandra J. Facey

Biogenesis of bacterial inner-membrane proteins

All cells must traffic proteins into and across their membranes. In bacteria, several pathways have evolved to enable protein transfer across the inner membrane, the periplasm, and the outer membrane. The major route of protein translocation in and across the cytoplasmic membrane is the general secretion pathway (Sec-pathway). The biogenesis of membrane proteins not only requires protein translocation but also coordinated targeting to the membrane beforehand and folding and assembly into their protein complexes afterwards to function properly in the cell. All these processes are responsible for the biogenesis of membrane proteins that mediate essential functions of the cell such as selective transport, energy conversion, cell division, extracellular signal sensing, and motility. This review will highlight the most recent developments on the structure and function of bacterial membrane proteins, focusing on the journey that integral membrane proteins take to find their final destination in the inner membrane.

The Cytoplasmic C-Terminal Domain of the Escherichia coli KdpD Protein Functions as a K+ Sensor

The KdpD protein is a K(+) sensor kinase located in the cytoplasmic membrane of Escherichia coli. It contains four transmembrane stretches and two short periplasmic loops of 4 and 10 amino acid residues, respectively. To determine which part of KdpD functions as a K(+) sensor, genetic variants were constructed with truncations or altered arrangements of the transmembrane segments. All KdpD constructs were tested by complementation of an E. coli kdpD deletion strain for their ability to grow at a K(+) concentration of 0.1 mM in the medium. A soluble protein composed of the C-terminal cytoplasmic domain was able to complement the kdpD deletion strain. In addition, analysis of the beta-galactosidase activity of an E. coli strain which carries a transcriptional fusion of the upstream region of the kdpFABC operon and a promoterless lacZ gene revealed that this soluble KdpD mutant responds to changes in the K(+) concentration in the extracellular medium. The results suggest that the sensing and response functions are both located in the C-terminal domain and might be modulated by the N-terminal domain as well as by membrane anchoring.

Membrane Protein Insertion of Variant MscL Proteins Occurs at YidC and SecYEG of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously.

An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane

The sensor protein KdpD of Escherichia coli is composed of a large N‐terminal hydrophilic region (aa 1–400), four transmembrane regions (aa 401–498) and a large hydrophilic region (aa 499–894) at the C‐terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP‐driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull‐down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino‐terminal domain of KdpD (aa 22–48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N‐terminal region in the cytoplasm can be membrane‐targeted early on to allow co‐translational membrane insertion of their distant transmembrane regions.

Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption

Saccharomyces cerevisiae does not express any xylose‐specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed.

Adsorption and Self-Assembly of M13 Phage into Directionally Organized Structures on C and SiO2 Films

A versatile method for the directional assembly of M13 phage using amorphous carbon and SiO2 thin films was demonstrated. A high affinity of the M13 phage macromolecules for incorporation into aligned structures on an amorphous carbon surface was observed at the concentration range, in which the viral nanofibers tend to disorder. In contrast, the viral particles showed less freedom to adopt an aligned orientation on SiO2 films when deposited in close vicinity. Here an interpretation of the role of the carbon surface in significant enhancement of adsorption and generation of viral arrays with a high orientational order was proposed in terms of surface chemistry and competitive electrostatic interactions. This study suggests the use of amorphous carbon substrates as a template for directional organization of a closely-packed and two-dimensional M13 viral film, which can be a promising route to mineralize a variety of smooth and homogeneous inorganic nanostructure layers.

Selection of peptides binding to metallic borides by screening M13 phage display libraries

BackgroundMetal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.ResultsIn this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.ConclusionsThis study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

Accelerated Nucleation of Hydroxyapatite Using an Engineered Hydrophobin Fusion Protein

Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.

Phage-assisted assembly of organic–inorganic hybrid bilayers

Abstract Protein-based bottom-up synthesis of functional nanomaterials and devices is one of the most promising areas in bio-nanotechnology. Here, we demonstrate that organic assemblies can serve as biologically controllable scaffolds for the deposition of inorganic nanoparticles. In this work, wild-type M13 phages were employed for controlled mineralization of zinc oxide particles. Our aim was to construct layered structures of organic and inorganic materials which contain alternating layers on a smooth substrate. The structure, elemental composition, and also the integrity of the organic and the biologically-templated inorganic layers were studied. A uniform nano-hybrid structure without significant thickness fluctuations was fabricated by using a high concentration of M13 phages and a carbon-coated substrate. The current study gives insight into the combination of organic–inorganic materials to form a multilayered structure, which in turn sets the stage for the fabrication of electronic devices, e. g. actuators or capacitors.

Draft Genome Sequence of Phenylobacterium immobile Strain E (DSM 1986), Isolated from Uncontaminated Soil in Ecuador

ABSTRACT We report the draft genome sequence of 3.3 Mb and the sequence (19.2 kb) of a natural plasmid isolated from Phenylobacterium immobile strain E (DSM 1986), able to degrade xenobiotic compounds as the sole carbon source. The sequences reveal a large number of novel Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs).